Studies on structural design, chemical synthesis, conformational properties and the mode of action of vertebrate peptide antibiotics, magainin are the focus of this program. We prepared all D amino acid analogues of our most active magainin peptides by the solid-phase method. The peptides designated as Endin A, B, F and G gave identical elution characteristics in reverse phase HPLC and mirror images in the CD spectra to their counterparts. Endin F as a representative was completely resistant to pronase digestion under conditions in which magainin F was totally degraded. The Endins were only 2-fold more active antibacterially. No difference in the spectrum of biological activity was observed. These results suggest that the proteolytic enzyme susceptibility has limited effect toward the increase of antibacterial activity. As it is expected, all D amino acid substituted analogues will exhibit in left handed helical form the same relationship between hydrophobic and hydrophilic faces as is observed in the L peptides. Thus, it appears that the helical amphiphilic conformation to provide correct interaction with lipid membrane is important in determining biological activities. Moreover, the mode of action of these peptides is unlikely to involve a stereospecific entity such as receptor or enzyme directly. The in vitro activities of magainin B, G and H against pathogenic protozoa Blastocystis hominis, Entamoeba histolytica and Trypanosoma cruzi were assessed for their morphological integrity and motility. The antiprotozoan activities were B>G>H the same order as the antibacterial activity. The parallelism between these two activities observed in these three analogues suggest that the helix structure of magainins is important in eliciting lysis activity on protozoa. Studies were also performed with seven highly potent all L-form analogues and seven D-Ala permuted analogues for their histamine-release (HR) activity in rat peritoneal mast cells. Magainins and analogues yielded low to moderate activity. In three duplicate experiments, the results have indicated consistently an inverse relationship between antimicrobial and the HR activity. Therefore, it may suggest that an alpha-helical conformation is not only unimportant but also unfavorable and the formation of anion channels is not necessary to achieve HR from mast cells. These studies indicate that magainin analogues not only have a practical therapeutic value in the treatment of infection and diseases in man and animals but also provide a model to correlate structure and thermodynamic variables with biological activities, and to understand unique properties related to peptide-phospholipid interaction.